CN108802210A - Injury of testis relevant difference opposite sex endogenous marker and its screening technique and application - Google Patents
Injury of testis relevant difference opposite sex endogenous marker and its screening technique and application Download PDFInfo
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Abstract
The invention discloses a kind of injury of testis biomarkers, are the compositions of the othernesses endogenous markers such as docosahexaenoic acid (Docosahexaenoic acid), linoleic acid (Linoleic acid), L- acetylcarnitines (L-Acetylcarnitine) or palmitic acid (Palmitic acid).The screening technique for the application and injury of testis marker that the present invention also provides the injury of testis mark compositions in preparing injury of testis detection reagent.The method of the present invention can filter out the injury of testis biomarker with stronger sensitivity and specificity, the marker can show significant difference in the case where obviously damage not yet occurs in testis tissue pathology, can preferably be used for the prevention and discovery of injury of testis.
Description
Technical field
The present invention relates to a kind of endogenous metabolism marker, more particularly to one kind are endogenous with the relevant otherness of injury of testis
Property marker and its screening technique and the application in detecting injury of testis.
Background technology
Testis is the main reproductive organs of male, and the major function of testis is the growth of Spermatogenic action and stimulation reproductive organs
Development etc., in recent years, the rising year by year of disease in the male sexual system incidence is primarily due to testis and has become toxic substance
The first target organs of matter damage.It is a hidden and long-term process since the function of testis is impaired, only occurs pathology and life
The variation of reason could judge that testis is damaged, especially embryonic development or gonad development foetal period, testicualr development period by
To interference, male reproductive function exception is directly contributed, but the research side of injury of testis is found due to lacking specificity and sensibility
Method, at present there is an urgent need for we establish one kind can early stage, accurate injury of testis evaluation method, to make up existing detection method
It is insufficient.
Metabolism group is to study the biochemical effect of organism own physiological pathological state and organism exogenous substance
Powerful measure, it is it is contemplated that metabolic pathway middle-molecular-weihydroxyethyl is less than the variation of 1000 small molecule metabolites.Metabolism group is answered
The change of endogenous metabolism object is detected with platforms such as nuclear magnetic resonance, liquid chromatography mass combination and gas chromatography combined with mass spectrometry
It can emphasize toxic effect mechanism across extensive biochemical pathway and identify the potential source biomolecule marker of testicular toxicity.
Therefore, it is necessary to find injury of testis biology using metabonomic technology, make the more existing inspection of the discovery of injury of testis
Survey method shifts to an earlier date, and can be preferably applied to the prevention and discovery of injury of testis, can shift to an earlier date into related treatment, make up existing
The deficiency of detection method.
Invention content
It is an object of the present invention to：A kind of injury of testis biomarker is provided, it can be effective for injury of testis
Prevention and discovery.
It is another object of the present invention to：The injury of testis mark compositions are provided and are preparing injury of testis detection
Application in reagent.
Another object of the present invention is：A kind of method and its screening model of screening injury of testis marker are provided
Method for building up can filter out the injury of testis biomarker with stronger sensitivity and specificity.
The present invention realizes that the scheme of above-mentioned purpose is as follows：
First, the present invention provides a kind of injury of testis biomarker, it is arbitrary two or more in following metabolin
Composition：Docosahexaenoic acid (Docosahexaenoic acid), linoleic acid (Linoleic acid), L- acetyl meat
Alkali (L-Acetylcarnitine) or palmitic acid (Palmitic acid)；Specifically composition can be：
The composition of docosahexaenoic acid (Docosahexaenoic acid) and linoleic acid (Linoleic acid),
Docosahexaenoic acid (Docosahexaenoic acid) and L- acetylcarnitines (L-Acetylcarnitine)
Composition,
The composition of docosahexaenoic acid (Docosahexaenoic acid) and palmitic acid (Palmitic acid),
The composition of linoleic acid (Linoleic acid) and L- acetylcarnitines (L-Acetylcarnitine),
The composition of L- acetylcarnitines (L-Acetylcarnitine) and palmitic acid (Palmitic acid),
The composition of linoleic acid (Linoleic acid) and palmitic acid (Palmitic acid),
Docosahexaenoic acid (Docosahexaenoic acid), linoleic acid (Linoleic acid) and L- acetyl meat
The composition of alkali (L-Acetylcarnitine),
Docosahexaenoic acid (Docosahexaenoic acid), L- acetylcarnitines (L-Acetylcarnitine) and
The composition of the composition of palmitic acid (Palmitic acid),
Linoleic acid (Linoleic acid), L- acetylcarnitines (L-Acetylcarnitine) and palmitic acid (Palmitic
Acid composition),
Docosahexaenoic acid (Docosahexaenoic acid), linoleic acid (Linoleic acid) and palmitic acid
The composition of (Palmitic acid),
Alternatively,
Docosahexaenoic acid (Docosahexaenoic acid), linoleic acid (Linoleic acid), L- acetylcarnitines
(L-Acetylcarnitine) and the composition of palmitic acid (Palmitic acid).
Currently preferred injury of testis biomarker further comprises elaidic acid (Elaidic acid) and/or figured silk fabrics
Propylhomoserin (Valine).
The preferred injury of testis biomarker of the present invention, further include following metabolin in any one or
Two or more compositions：Stearic acid (Stearic acid), lactic acid (Lactic acid), leucine (Leucine), amber
Sour (Succinic acid) or leukotrienes (Linolenic Acid), eicosapentaenoic acid (Eicosapentaenoic
Acid), arachidonic acid (Arachidonic acid), erucic acid (Erucic acid), creatine (Creatine), spermin
(Spermin) or taurine (Taurine).
The most preferably described injury of testis biomarker of the present invention, is docosahexaenoic acid (Docosahexaenoic
Acid), linoleic acid (Linoleic acid), L- acetylcarnitines (L-Acetylcarnitine), palmitic acid (Palmitic
Acid), elaidic acid (Elaidic acid), valine (Valine), stearic acid (Stearic acid), lactic acid (Lactic
Acid), leucine (Leucine), succinic acid (Succinic acid), leukotrienes (Linolenic Acid), 20 light dydrocarbons
Olefin(e) acid (Eicosapentaenoic acid), arachidonic acid (Arachidonic acid), erucic acid (Erucic acid),
The composition of creatine (Creatine), spermin (Spermin) and taurine (Taurine).
The present invention further provides application of the biomarker in the kit for preparing detection injury of testis；Institute
The application stated is to prepare the kit of detection injury of testis using biomarker of the present invention as detection target.
The present invention also provides a kind of kits of detection injury of testis, wherein containing being useful for detecting biology of the present invention
The reagent of marker.
The present invention also provides a kind of injury of testis biomarker screening technique, be using high resolution mass spectrum foundation include fat
The database of fat class, carbohydrate, steroids, neurotransmitter and visceral organ injury related substances establishes the inspection of all kinds of endogenous materials
Survey method is finally utilized using the endogenous material in Tracefounder software qualitative analysis testis samples
The difference of MetaboAnalyst3.0 web analytics testis endogenous metabolism objects draws PCA and PLS-DA illustratons of model, according to VIP
>1 selects the endogenous metabolism object with significant difference, the significant endogenous metabolism object as injury of testis.
Screening technique of the present invention specifically includes sample pre-treatments, data acquisition, endogenous metabolism object are identified, more
First Random data processing；Steps are as follows：
1) testis sample is conventionally handled, LC-MS/MS is complied with2Analyze sampling condition；
2) by step 1), treated that sample is LC-MS/MS2Analysis is completed the acquisition of metabolite profile diagram data, is metabolized
Object chromatographic retention and mass-to-charge ratio；Wherein,
It is using feature chromatographic condition：Chromatographic condition：A：Water (2mmoL/L ammonium formates), D：Acetonitrile；Gradient elution, gradient
Elution program:0-1.0min, 5%D；1.0-5.0min, 5%-60%D；5.0-8.0min, 60%-100%D；8.0-
11.0min 100%D；11.0-14.0min 100%-60%D；14.0-15.0min 60%-5%D；15.0-18.0min,
5%D, 0~18min of analysis time, sample size 5 μ L, flow velocity 0.25mL/min, chromatographic column：1.7 μm of ACQUITY BEH C18,
2.1×50mm；
It is using characterising mass spectrometry condition：Ion source：ESI (±), source parameters：Spray voltage：2800V；Evaporating temperature：
350℃；Sheath gas：35Arb；Assist gas：10Arb；Capillary temperature：350℃；S-lens RF：50；Chemical parameters：Level-one
Full scan (Full scan)：Resolution ratio：70000；AGC target：1e6；Maximun TT：100ms；Scanning range： 70-
1050m/z；
3) it is directed to the identification that the metabolite profile data that step 2) obtains carry out endogenous metabolism object, establishes endogenous metabolism object
Database；
4) database for the endogenous metabolism object established to step 3) carries out multivariate statistics data processing, finds out injury of testis
Whether metabolic patterns difference and apparent classification trend, finally conventionally carry out difference metabolin screening.
In screening technique of the present invention, the identification of the endogenous metabolism object described in step 3) is preferably by high-resolution matter
Spectrum mzCloud obtains the accurate mass number of the decimal point 5 of each endogenous metabolism object, and passes through each endogenous metabolism
The molecular formula of object identifies, Tracefinder softwares is recycled to establish the database of endogenous metabolism object.
In screening technique of the present invention, multivariate statistics data processing described in step 4) preferably by
The peak area for the corresponding endogenous metabolism object of database automatic collection that Tracefinder softwares are established recycles
MetaboAnalyst3.0 web analytics, draw PCA and PLS-DA illustratons of model, and metabolic patterns whether finding out injury of testis are poor
Different and apparent classification trend, finally according to VIP>1 standard selects the endogenous metabolism object with significant difference.
The present invention also provides the method for building up that a kind of injury of testis early diagnoses model, specifically include sample pre-treatments, number
According to acquisition, the identification of endogenous metabolism object, multivariate statistics data processing；Steps are as follows：
1) no less than 100 testis tissue samples are collected, each sample is conventionally handled to meeting LC-MS/
MS2Analyze sampling condition；
2) by step 1), treated that sample is LC-MS/MS2Analysis is completed the acquisition of metabolite profile diagram data, is metabolized
Object chromatographic retention and mass-to-charge ratio；Wherein,
It is using feature chromatographic condition：Chromatographic condition：A：Water (2mmoL/L ammonium formates), D：Acetonitrile；Gradient elution, gradient
Elution program:0-1.0min, 5%D；1.0-5.0min, 5%-60%D；5.0-8.0min, 60%-100%D；8.0-
11.0min 100%D；11.0-14.0min 100%-60%D；14.0-15.0min 60%-5%D；15.0-18.0min,
5%D, 0~18min of analysis time, sample size 5 μ L, flow velocity 0.25mL/min, chromatographic column：1.7 μm of ACQUITY BEH C18,
2.1×50mm；
It is using characterising mass spectrometry condition：Ion source：ESI (±), source parameters：Spray voltage：2800V；Evaporating temperature：
350℃；Sheath gas：35Arb；Assist gas：10Arb；Capillary temperature：350℃；S-lens RF：50；Chemical parameters：Level-one
Full scan (Full scan)：Resolution ratio：70000；AGC target：1e6；Maximun TT：100ms；Scanning range： 70-
1050m/z；
3) it is directed to the identification that the metabolite profile data that step 2) obtains carry out endogenous metabolism object, establishes endogenous metabolism object
Database；
4) database for the endogenous metabolism object established to step 3) carries out multivariate statistics data processing, and it is normal to find out testis
Sample and injury of testis sample metabolic patterns difference and apparent classification trend, it is poor in injury of testis sample to filter out
Anisotropic endogenous metabolism object is as injury of testis marker, and the data based on these markers are established injury of testis early stage and examined
Disconnected model.
In method for establishing model of the present invention, the identification of the endogenous metabolism object described in step 3) is preferably by high score
It distinguishes that mass spectrum mzCloud obtains the accurate mass number of the decimal point 5 of each endogenous metabolism object, and passes through each endogenous
The molecular formula of metabolin identifies, Tracefinder softwares is recycled to establish the database of endogenous metabolism object.
In method for establishing model of the present invention, multivariate statistics data processing described in step 4) preferably by
The peak area for the corresponding endogenous metabolism object of database automatic collection that Tracefinder softwares are established recycles
MetaboAnalyst3.0 web analytics, draw PCA and PLS-DA illustratons of model, and metabolic patterns whether finding out injury of testis are poor
Different and apparent classification trend, according to VIP>1 standard selects the endogenous metabolism object with significant difference, is based ultimately upon
These metabolins establish injury of testis early diagnosis model.
Compared with prior art, the present invention makes to adopt by optimizing feature chromatography and characterising mass spectrometry condition in data acquisition
The sample data collected is more accurate, lays the foundation for further screening otherness endogenous material, in conjunction with high-resolution matter
The characteristics of sensitivity of spectrum and negative ions full scan, makes the injury of testis biomarker filtered out have stronger sensitive
Property and specificity, they can testis tissue pathology not yet occur obviously damage in the case of show significant difference, energy
Enough preventions and discovery for being preferably used for injury of testis.
Description of the drawings
Figure 1A is zoopery high resolution mass spectrum detection gained cation full scan total ion current figure in embodiment 1.
Figure 1B is zoopery high resolution mass spectrum detection gained anion full scan total ion current figure in embodiment 1.
Fig. 1 C are zoopery high resolution mass spectrum detection gained valine chromatograms in embodiment 1.
Fig. 1 D are the corresponding accurate mass number of zoopery high resolution mass spectrum detection gained valine and molecule in embodiment 1
Formula.
Fig. 2 a are the PCA shot charts of each contamination group multi-variate statistical analysis of zoopery injury of testis in embodiment 1.
Fig. 2 b are the PLS-DA three-dimensional scores of each contamination group multi-variate statistical analysis of zoopery injury of testis in embodiment 1
Figure.
Fig. 3 A are zoopery Normal group testis tissue slice maps in embodiment 1.
Fig. 3 B are zoopery DEHP groups testis tissue slice maps in embodiment 1.
Specific implementation mode
Unless specifically defined, present invention description term used is the well known term in related technical field.Standard
Chemical symbol and dummy suffix notation can be used interchangeably with its full name.
It is indicated except no special, the present invention is used but not yet explicitly elaboration or the technology and methods simply illustrated refer to this technology
The usually used technology and methods in field can be carried out according to technology and methods well known in the art.The use of kit is basis
The specification that manufacturer or supplier provide carries out.
Embodiment 1.
A kind of screening injury of testis biomarker screening technique, by taking the screening of experimental animal as an example, scheme is as follows：
1. reagent：DEHP is purchased from Sigma-Aldrich companies, and 75% ethyl alcohol is purchased from one factory of Shanghai development chemical industry, chromatographic grade
Acetonitrile, methanol and formic acid are purchased from Sigma-Aldrich companies.
2. zoopery：Male 5-6w old C57BL/6 mouse routine feedings, free water, receptacle light 10h are black
Dark 14h, 21-25 DEG C of temperature, humidity 30%-70%, 24 mouse are randomly divided into 2 groups, every group 12, respectively normal control
Group and 500mg/kg gavage contaminations group (DEHP groups), Normal group gives isometric solvent, 1 time a day, continuous 35 days.
After gavage 35 days, mouse, with full automatic biochemical apparatus detection biochemical indicator (see the table below 1), is then led to by taking blood after etherization
It crosses carbon dioxide inhalation and puts to death mouse, take out testis, and testicular weight of weighing, record organ coefficient (see the table below 2).Part
10% dipped into formalin of testis is used for pathological section, and remaining testis, which freezes, to be detected in liquid nitrogen for metabolism group.
Table 1
Table 2
3. the high resolution mass spectrum of testis sample detects
Chromatographic condition：A：Water (2mmoL/L ammonium formates), D：Acetonitrile；Gradient elution, gradient elution program:0-1.0min,
5% D；1.0-5.0min, 5%-60%D；5.0-8.0min, 60%-100%D；8.0-11.0min, 100%D；11.0-
14.0min 100%-60%D；14.0-15.0min 60%-5%D；15.0-18.0min, 5%D, analysis time 0~
18min, sample size 5 μ L, flow velocity 0.25mL/min, chromatographic column：1.7 μm of ACQUITY BEH C18,2.1 × 50mm.Mass spectrum item
Part：Ion source：ESI (±), source parameters：Spray voltage：2800V；Evaporating temperature：350℃；Sheath gas：35Arb；Auxiliary
Gas：10Arb；Capillary temperature：350℃；S-lens RF：50.Chemical parameters：Level-one full scan (Full scan)：It differentiates
Rate：70000； AGC target：1e6；Maximun TT：100ms；Scanning range：70-1050m/z.Mouse testis sample is used
Ultra-pure water (containing 50% acetonitrile) presses 1:10 mass volume ratio is homogenized, and takes 50 μ L of homogenate, (the methanol of containing the internal standard is added:
Acetonitrile=1:1) 450 μ L of precipitating reagent, vortex mixing 60s, 13000rpm centrifuge 10min, draw 5 μ L and be measured.In cation
It is designated as Propranolol, orinase is designated as in anion.Measure cation full scan total ion current figure (Figure 1A), bear from
Sub- full scan total ion current figure (Figure 1B), valine chromatogram (Fig. 1 C) and the corresponding accurate mass number of valine and molecular formula
(Fig. 1 D).
4. data analysis：The database automatic collection corresponding endogenous metabolism object established using Tracefinder softwares
Peak area recycles MetaboAnalyst3.0 web analytics, draws PCA shot charts (Fig. 2 a) and PLS-DA illustratons of model (figure
2b), it can be seen that control group from Fig. 2 a, 2b and DEHP groups can be separated clearly, illustrate two groups of experimental animals in metabolic patterns
On show significant difference and embody classification trend.
Finally according to VIP>1 standard selects the following endogenous metabolism object (being shown in Table 5) with significant difference.
Table 5
By in upper table as it can be seen that the endogenous metabolism for screening to obtain 17 kinds with injury of testis according to the screening technique of the present embodiment
Marker, existing research have confirmed that a variety of metabolic pathways of these metabolins and testis are closely related, and the VIP values the big right
Answer the probability of injury of testis bigger, in 17 kinds of markers, docosahexaenoic acid, linoleic acid, L- acetylcarnitines and palmitic acid
VIP values are up to 4.9 or more, belong to the metabolic markers highly relevant with injury of testis, the VIP values of remaining marker are also all
Higher than 1.5, good mark can be played the role of to the damage of testis.
Pathological study：Mouse takes the testis rinsed well to be put into 10% neutrality Fu Er after being put to death using carbon dioxide inhalation
It is fixed in Malin's solution, it repaiies cut through tissue successively, paraffin embedding, conventional section, HE dyeing, alcohol serial dehydration is transparent, envelope
Piece carries out light microscope pathological study, and pathology slice is as shown in Figure 3A and Figure 3B, and wherein Fig. 3 A are embodied normally
The testis tissue microstructure of control group, Fig. 3 B embody DEHP group testis tissue microstructures, and the instruction of C arrows is smart female
Cell, the instruction of D arrows is spermatoblast.By Fig. 3 A and Fig. 3 B compare it can be seen from sperm mother cell in DEHP group mouse testis
It is significantly reduced with sperm quantity, demonstrates the generation of injury of testis.
Claims (10)
1. a kind of injury of testis biomarker, it is the arbitrary two or more composition in following metabolin：22 carbon
Acid (Docosahexaenoic acid), linoleic acid (Linoleic acid), L- acetylcarnitines (L-
) or palmitic acid (Palmitic acid) Acetylcarnitine.
2. injury of testis biomarker described in claim 1, it is any one in following composition：
The composition of docosahexaenoic acid (Docosahexaenoic acid) and linoleic acid (Linoleic acid),
The combination of docosahexaenoic acid (Docosahexaenoic acid) and L- acetylcarnitines (L-Acetylcarnitine)
Object,
The composition of docosahexaenoic acid (Docosahexaenoic acid) and palmitic acid (Palmitic acid),
The composition of linoleic acid (Linoleic acid) and L- acetylcarnitines (L-Acetylcarnitine),
The composition of L- acetylcarnitines (L-Acetylcarnitine) and palmitic acid (Palmitic acid),
The composition of linoleic acid (Linoleic acid) and palmitic acid (Palmitic acid),
Docosahexaenoic acid (Docosahexaenoic acid), linoleic acid (Linoleic acid) and L- acetylcarnitines (L-
Acetylcarnitine composition),
Docosahexaenoic acid (Docosahexaenoic acid), L- acetylcarnitines (L-Acetylcarnitine) and palm
The composition of the composition of sour (Palmitic acid),
Linoleic acid (Linoleic acid), L- acetylcarnitines (L-Acetylcarnitine) and palmitic acid (Palmitic
Acid composition),
Docosahexaenoic acid (Docosahexaenoic acid), linoleic acid (Linoleic acid) and palmitic acid
The composition of (Palmitic acid),
Alternatively,
Docosahexaenoic acid (Docosahexaenoic acid), linoleic acid (Linoleic acid), L- acetylcarnitines (L-
) and the composition of palmitic acid (Palmitic acid) Acetylcarnitine.
3. the injury of testis biomarker described in claims 1 or 2 any one, further comprises elaidic acid (Elaidic
) and/or valine (Valine) acid.
4. the injury of testis biomarker described in claim 3, further include in following metabolin any one or
Two or more compositions：Stearic acid (Stearic acid), lactic acid (Lactic acid), leucine (Leucine), amber
Sour (Succinic acid) or leukotrienes (Linolenic Acid), eicosapentaenoic acid (Eicosapentaenoic
Acid), arachidonic acid (Arachidonic acid), erucic acid (Erucic acid), creatine (Creatine), spermin
(Spermin) or taurine (Taurine).
5. injury of testis biomarker described in claim 1 is docosahexaenoic acid (Docosahexaenoic
Acid), linoleic acid (Linoleic acid), L- acetylcarnitines (L-Acetylcarnitine), palmitic acid (Palmitic
Acid), elaidic acid (Elaidic acid), valine (Valine), stearic acid (Stearic acid), lactic acid (Lactic
Acid), leucine (Leucine), succinic acid (Succinic acid), leukotrienes (Linolenic Acid), eicosapentaenoic
Sour (Eicosapentaenoic acid), arachidonic acid (Arachidonic acid), erucic acid (Erucic acid), creatine
(Creatine), the composition of spermin (Spermin) and taurine (Taurine).
6. application of the biomarker described in claim 1 in the kit for preparing detection injury of testis, it is characterised in that：
The biomarker is prepared to the kit of detection injury of testis as detection target.
7. the kit of injury of testis is detected, wherein containing the reagent for being useful for the biomarker described in test right requirement 1.
8. a kind of screening technique of injury of testis biomarker specifically includes sample pre-treatments, data acquisition, endogenous metabolism
Identification, the multivariate statistics data processing of object；Steps are as follows：
1) testis sample is conventionally handled, LC-MS/MS is complied with2Analyze sampling condition；
2) by step 1), treated that sample is LC-MS/MS2The acquisition of metabolite profile diagram data is completed in analysis, is obtained metabolism and is looked for
Compose retention time and mass-to-charge ratio；Wherein,
It is using feature chromatographic condition：Chromatographic condition：A：Water (2mmoL/L ammonium formates), D：Acetonitrile；Gradient elution, gradient elution
Program:0-1.0min, 5%D；1.0-5.0min, 5%-60%D；5.0-8.0min, 60%-100%D；8.0-11.0min,
100%D；11.0-14.0min 100%-60%D；14.0-15.0min 60%-5%D；15.0-18.0min 5%D, analysis
0~18min of time, sample size 5 μ L, flow velocity 0.25mL/min, chromatographic column：1.7 μm of ACQUITY BEH C18,2.1 × 50mm；
It is using characterising mass spectrometry condition：Ion source：ESI (±), source parameters：Spray voltage：2800V；Evaporating temperature：350
℃；Sheath gas：35Arb；Assist gas：10Arb；Capillary temperature：350℃；S-lens RF：50；Chemical parameters：Level-one is swept entirely
It retouches (Full scan)：Resolution ratio：70000；AGC target：1e6；Maximun TT：100ms；Scanning range：70-1050m/
z；
3) it is directed to the identification that the metabolite profile data that step 2) obtains carry out endogenous metabolism object, establishes the number of endogenous metabolism object
According to library；
4) database for the endogenous metabolism object established to step 3) carries out multivariate statistics data processing, whether finding out injury of testis
Metabolic patterns difference and apparent classification trend, finally conventionally carry out difference metabolin screening.
9. screening technique according to any one of claims 8, it is characterised in that：The identification and utilization of endogenous metabolism object described in step 3) is high
Resolution Mass Spectrometry mzCloud obtains the accurate mass number of the decimal point 5 of each endogenous metabolism object, and passes through each endogenous
The molecular formula of metabolin identifies, Tracefinder softwares is recycled to establish the database of endogenous metabolism object.
10. screening technique according to any one of claims 8, it is characterised in that：Multivariate statistics data processing described in step 4) utilizes
The peak area for the corresponding endogenous metabolism object of database automatic collection that Tracefinder softwares are established recycles
MetaboAnalyst3.0 web analytics draw PCA and PLS-DA illustratons of model, find out metabolic patterns difference whether injury of testis
With apparent classification trend, finally according to VIP>1 standard selects the endogenous metabolism object with significant difference.
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